A 1210477

Apogossypol and Leflunomide from ApexBio (Boston, MA, USA), A-1331852, A-1210477, ABT-199, Z-VAD.FMK and CCCP from Selleck (Houston, TX, USA), Teriflunomide, norhydroguaiaretic acid (NDGA), Ivermectin, Terfenadine, Suloctidil, orotate and uridine from Sigma Aldrich (St Louis, MO, USA), MitoTracker Deep Red FM from Thermo Fisher (Loughborough, UK) were used. Antibodies against BAP31, RTN4, BiP, PDI, CHOP, DHODH and tubulin from Abcam (Cambridge, UK), CLIMP-63 and BAX (6A7) from Enzo Life Sciences (Exeter, UK), TIM22 and KNT-1 from Sigma, HSP60, Cytochrome c, BAX, OPA1 and DRP-1 from BD Biosciences (San Jose, CA, USA), phospho-DRP-1 (S616), phospho-DRP-1 (S637), MFN1 and MFN2 from Cell Signaling Technologies (Danvers, MA, USA), BAK (AB-1) from Calbiochem (Watford, UK), MFF, MID49 and MID51 from ProteinTech (Manchester, UK) and GAPDH from Santa Cruz Biotechnologies (Santa Cruz, CA, USA) were used. For RNA interference, cells were transfected with 10 nM of siRNAs against DHODH (SI00363384 and SI00363391) purchased from Qiagen Ltd. (Manchester, UK), using Interferin (Polyplus Transfection Inc, NY), according to the manufacturer’s protocol and processed 72 h after transfection.

UMI-77 (APExBIO);^{8 (link)} Bax-inhibiting peptide V5 (BIP-V5; Sigma Aldrich); MG132 (Enzo Life Sciences), etoposide (Cell Signaling Technologies); PAC1, Bam7, Birinapant, Lexibulin (CYT997), Nutlin-3a, YM155, A1210477, Navitoclax (ABT-263), Venetoclax (ABT-199) and Obatoclax mesylate were all purchased from Selleckchem. All inhibitors and stressors were dissolved in DMSO; control conditions were all treated with the appropriate amount of DMSO.

ABT199, ABT263 and A-1210477 were purchased from Selleck Chemicals while QVD-OPH was purchased from Sigmaaldrich. For in vivo studies ABT199 and ABT263 were formulated in 10% ethanol/30% polyethylene glycol (PEG) 400/60% phosal 50 propylene glycol (PG) (v/v/v) in final concentrations of 10 mg/mL.

HeLa cells were seeded the day before transfection. siRNA (20 nM final concentration) was mixed with Lipofectamin RNAiMAX (Thermo Scientific) at 1:0.83 v/v in serum-free medium (Optimem, Thermo Scientific), incubated for 20 min at RT and added to the cells. siRNA specific for Mcl-1 (GGCAGTCGCTGGAGATTAT) or scrambled siRNA (termed siCtrl; negative control low GC, #12935-200 Thermo Scientific) was used. Alternatively, the specific Mcl-1 inhibitor A-1210477 (#S7790, Selleck Chemicals, Munich, Germany) was added to the cells (10 μM) after infection with MVA.

Authenticated stocks of cell line U-2946 were grown in RPMI 1640 (Invitrogen, Darmstadt, Germany) containing 10% fetal bovine serum (FBS) (Sigma-Aldrich, Taufkirchen, Germany). Cell lines applied in this study are all held by the DSMZ—German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany (www.dsmz.de) or were supplied by the originators for research purpose. Detailed references and cultivation protocols have been described previously [19 ]. The BCL2 family inhibitors ABT-263 and A-1210477 were obtained from Selleckchem (München, Germany).

ABT-199, A-1210477 and Z-VAD.FMK from Selleck (Houston, TX, USA), S63845 from Active Biochem (Kowloon, Hong Kong) and A-1331852 from AbbVie Inc. (North Chicago, IL, USA) were used. Antibodies against HSP70 (cat#ab2799) from Abcam (Cambridge, UK), OPA1 (cat#612607), cytochrome c (cat#556432) and DRP-1 (cat#611113) from BD Biosciences (San Jose, CA, USA); BCL-X_L (cat#2762), BIM (cat#2933) and BAD (cat#9292) from Cell Signalling Technology (MA, USA); BAK (AB-1) (cat#AM-03) and NOXA (cat#OP180) from Millipore (Watford, UK) and MCL-1 (cat#sc-819), BAK (cat#sc-832) and GAPDH (cat#sc-25778) from Santa Cruz Biotechnologies (Santa Cruz, CA, USA) were used. All other reagents were obtained from Sigma Aldrich (St. Louis, MO, USA).

Pools of BIMs2A or empty vector (EV) cells were made by routine cloning into an all-in-one tetracycline inducible vector (SH570MK as detailed in Additional file 1) and selected using Puromycin. BIMs2A expression was induced with 2 μg/ml DOX or vehicle control daily in the media and cells were harvested at the time points indicated in the figures. Annexin V PI staining was performed using the Annexin V-FITC Apoptosis Kit (Biovision, CA, USA) as per the manufacturer’s instructions. ABT-263 (5 μg/ml) was added to the media at the indicated times. For siRNA experiments, 5 nM siRNAs targeting MCL-1 (DHA-L-004384-00-0005) or non-targeting control siRNA (D-001206-14-05) was premixed with RNAiMAX (ThermoFisher) and cells were transfected the day after plating at a density of 1 × 10⁵ cells per well with ON-TARGETplus SmartPools of MCL-1 or nontargeting controls siRNAs as per the manufacturer’s instructions. A1210477 and UMI-77 (Selleckchem, MA, USA) was added to the media at 5 or 10 μM respectively as indicated in the figures. Dasatinib (Bristol-Myers Squibb, Princeton, NJ, USA) was added to the media in 2D and 3D at a concentration of 1 μM and 200 nM respectively. 3D collagen I/fibroblast models were performed as described previously [20 ].